Methods and compositions for the inhibition of growth of infectious Aspergillus fumigatus and other mycotic organisms in the gut of mammalian and avian species

ABSTRACT

A method for the prevention and treatment of fungal infections and, therefore, consequent invasive mycosis in mammalian and avian species is described. The invention comprises a combination of β-1,3(4)-endoglucanohydrolase, β-1,3(4)glucan, diatornaceous earth, mineral clay, and glucomannan, which is fed to or consumed by mammalian or avian species in amounts sufficient to inhibit enteric fungal colonization in the gut and consequent mycosis. The invention described may be admixed with feeds or foods, incorporated into pelleted feeds or foods or administered orally to mammalian and avian species.

REFERENCES CITED [REFERENCED BY]

U.S. Patent Documents 3961080 October, 1973 Sugimoto et al. 426/605165946 July, 1990 Taylor 426/74 5149549 September, 1992 Beggs 426/25192547 October, 1990 Taylor 424/438 5639492 January, 1995 Turk 426/25814346 June, 1996 Gamberini 426/665 5935623 January, 1998 Alonso-Debolt426/2 5988599 March, 1996 Subbiah 514/703 6045834 April, 1999 Howes andNewman 426/2 6221381 April, 2001 Shelford and Kamande 424/442 6344221May, 2000 Evans 426/2 6476003 November, 2002 Jordan et al. 514/54

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BACKGROUND OF THE INVENTION

1. Field of the Invention

Methods and compositions for the inhibition of growth of infectiousAspergillus fumigatus and other pathogenic mycotic organisms in thegastrointestinal tract of mammalian and avian species.

2. Background

Aspergillosis and mycotic infections. Aspergillosis is an acute systemicmycotic infection caused by Aspergillus sp. Species of Aspergillus knownto cause an infection in mammals and avian species include Aspergillusfumigatus, Aspergillus flavis and Aspergillus niger. Infectiousaspergillosis has been involved in mycotic abortions and relateddiseases (Jensen et al.,1991; McCausland et al., 1987). Medicalliterature contains numerous references indicating an increasingincidence of small bowel infarctions and coagulopathies in humansrelated to aspergillosis (Catalano et al., 1997; Prescott et al., 1992;Oshawa, 1991). Aspergillus fumigatus has also been implicated as apossible etiological agent in Jejunal Hemorrhage Syndrome in cattle, anew emerging disease that causes massive hemorrhaging of the smallintestine (Puntenney et al., 2003). This disease also affectsimmunocompromised humans. Aspergillosis is also documented to cause ahigh incidence of abortions and pneumonia in cattle, a source ofsignificant economic loss to the livestock industry (Puntenney et al.,2003).

Innoculation of an animal with Aspergillus sp. is commonly throughingestion of mold-contaminated feedstuffs due to improper storage orharvesting techniques, fecal contamination of feedstuffs from birds androdents, and inhalation of Aspergillus spores from bedding material. Theorganism is especially effective in evasion of host animal defenses, bysecretion of various lipid compounds, including fumigillin,fumitremorgin A, fumigaclavine, and gliotoxin at the infection site,impairing localized generation of complement factors C3a and C5a,thereby blocking recruitment of polymorphonuclear cells. Phagocyticcells normally follow the chemotactic gradient of complement componentsto the site of the infection, where engulfment and elimination of theorganism from the animal occurs (Rhodes et al., 1992). The ability tosequester iron is a virulence factor for microorganisms. The binding ofcirculating iron to transferrin and lactoferrin, as well as theintercellular storage of iron, reduce levels of free iron below thatrequired for microbial growth. Aspergillus sp. produces two majorsiderophores (high affinity iron binding compounds) that competesuccessfully with transferrin and lactoferrin to acquire iron forgrowth: N,N′,N″-triacetylfusarinine C and ferricrocin. Proteolyticdigestion of transferrin may also be utilized as a means of ironacquisition. Iron is released from transferrin at pH<6 as a result ofprotonation of the iron binding site generally localized at infectionsite (Rhodes et al.,1992).

Aspergillus sp. produces two serine proteinases, elastinolytic andazocollytic enzymes, which break down tissue (Tomee and Kauffman, 2000;Frosco et al. ,1989). Proteinase enzyme production is tied to tissueinvasiveness and facilitates entry from colonization site into theparenchyma. Hemorrhagic infarction (vascular blockage) and tissuenecrosis may follow infection (Rhodes et al., 1992). After tissueinvasion, dissemination via the bloodstream to other organs and theplacenta occurs. Placentitis and subsequent abortions commonly result.

RATIONAL DEVELOPMENT OF THE INVENTION

The fungal cell wall is composed of highly-branched β-1,3 glucans withlinkages to chitin, galactomannan and a linear β-1,3/1,4 glucan(Fontaine, et al., 2000). Understanding of the structural biology ofpathogenic fungi has aided the design of the invention. The efficacy ofthe invention is partly related to the addition of1-1,3(4)-endoglucanohydrolase, which delays initiation of fungal growthvia enzymatic degradation of cell wall components.

A second component of the invention, which functions in tandem withβ-1,3(4)-endoglucanohydrolase, is β-1,3 glucan. The role of β-1,3 glucanin macrophage activation and response to pathogens is well documented inthe literature (Czop, et al., 1985, Xia et al,1999). Upon entering theaqueous conditions of the digestive tract, the large glucan molecules inthe invention, via the enzymatic activity ofβ-1,3(4)-endoglucanohydrolase, are effectively reduced to smallermoieties that may be accepted by macrophage receptors, stimulatingimmunological activation. Other components of the invention,diatomaceous earth and aluminosilicate, are capable of binding toxiclipid compounds which are secreted by pathogenic fungi. These toxins, ifnot bound, effectively block release of complement factors required formacrophage response to colonization.

The feeding of a combination of β-1,3(4)-endoglucanohydrolase, β-glucan,calcined diatomaceous earth (hereafter identified as diatomaceous earth;SiO₂), glucomannan, and mineral clay, such as aluminum silicate,montmorillonite clay, bentonite or zeolite, has been shown to delaygrowth of A. fumigatus for a period of several hours in laboratorycultures and clinical trials thereby effectively minimizing the abilityof Aspergillus to inhibit complement factor production thereby allowingan intact immune system to respond to fungal colonization.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a novel and previouslyunknown method for inhibition of the colonization of the digestivetracts and consequent systemic infection (mycosis) of mammalian andavian species by various pathogenic microorganisms (specificallypathogenic fungal organisms). The invention may be applied to, but notlimited to, in situ inhibition of the enteric growth and mycosis causedby various pathogenic microorganisms including Aspergillus,Aureobasidium, Candida, Eupenicillium, Eurotium, Fusarium, Mucor,Penicillium, Rachihorskiomyces and other genera which comprise thefungal taxonomic classification (Kingdom Fungi) as outlined byAlexopolous et al., 1996.

A further object of this invention is to provide a method for inhibitionof pathogenic microbial growth in situ and, consequently, in blood andto thereby minimize or obviate morbidities and mortalities caused by,but not limited to, pathogenic fungi with a preparation comprising acombination of β-1,3(4)-endoglucanohydrolase, β-glucan, diatomaceousearth, glucomannan, and mineral clay, such as aluminum silicate,montmorillonite clay, bentonite or zeolite.

Another object of the invention is to provide a composition comprising acombination of β-1,3(4)-endoglucanohydrolase, β-glucan, diatomaceousearth, mineral clay, and glucomannan, which additively minimizes growthand, thereby, reduces mycotic potential of pathogenic fungi in thegastrointestinal tracts of mammalian and avian species.

Additional objects, advantages and novel features of the invention willbe set forth, in part, in the description that follows and will, inpart, become apparent to those skilled in the art upon examination ofthe following or may be learned with the practice of the invention. Toachieve the foregoing and other objects, and in accordance with thepurposes of the present invention as described herein, a novel method isdescribed for the inhibition of growth of pathogenic fungal organismswhich typically underlie enteric-based and mycotic morbidities andmortalities of mammalian and avian species. In particular, thisinvention minimizes or eliminates the colonization of thegastrointestinal tract by pathogenic fungi, reduces the populations ofpathogenic organisms in blood and thereby minimizes or eliminatespathologies directly caused by and indirectly caused by thiscolonization. The invention comprises a mixture ofβ-1,3(4)-endoglucanohydrolase, β-glucan, diatomaceous earth, mineralclay, and glucomannan. The diatomaceous earth is standard commercialgrade available from a variety of sources. Theβ-1,3(4)-endoglucanohydrolase is produced from submerged fermentation ofa strain of Trichoderma longibrachiatum. The β-1,3(4)glucan andglucomannan are derived from a commercial product and are an extractionfrom any of a number of yeast organisms. The mineral clay product is astandard commercial grade (examples include, but are not limited to,montmorillonite clay, bentonite and zeolite). Extractions andproductions of diatomaceous earth, yeast cell wall extract and mineralclay are well known in the art and commercially-available.

The compositions which are provided by the invention can be fed to anymammalian or avian species including, but not limited to, bovine,equine, ovine, caprine and avian species. When admixed with the feed orfood or fed as a supplement, the invention minimizes or eliminates thegrowth of pathogenic fungi in the gut thereby allowing colonization ofthe gut with non-pathogenic species. The invention also minimizes oreliminates invasion of the blood compartment by pathogenic fungi. Theinvention thereby minimizes or eliminates the manifestations of thepathologies typically associated with enteric fungal infections.Administration of the product may be used as a prophylactic (i.e., toprevent colonization and growth of pathogenic fungal species in the gutof mammalian or avian species), as an additive to feeds or foodsinfected with pathogenic fungi or as a preferred method to treat andthereby minimize or eliminate an existing, diagnosed or non-diagnosed,enteric fungal infection and mycoses. Application of the invention asdescribed herein and via the specific and novel mechanisms describedherein will minimize and possibly eliminate manifestations of entericfungal infections and, consequently, mycotic infections including, butnot limited to, the following genera: Aspergillus, Aureobasidium,Candida, Eurotium, Fusarium, Mucor, Penicillium and Rachiborskiomyces.Application of the invention as described herein will also minimize orpossibly eliminate manifestations associated with the presence ofpathogenic fungal organisms, as identified above, in food or feed ofmammalian and avian species.

DESCRIPTIONS OF THE DRAWINGS

The accompanying drawings and photographs which are incorporated intothe following “Detailed Description of the Invention” form part of thespecification and illustrate several aspects of the present inventionand, together with the Detailed Description, serve to explain thedetails of the invention. In the following section:

FIG. 1 shows the effects of adding 50 μl of a mixture of 95.6% mineralclay and 4.4% β-1,3(4)glucan and glucomannan extract (40 mg/ml) on thegrowth of Aspergillus fumigatus on a Sabouraud dextrose agar platesupplemented with chloramphenicol and gentimycin.

FIG. 2 shows the effects of adding a low concentration (100 μg/10 mlculture) of a mixture of 95.6% mineral clay and 4.4% β-1,3(4)glucan andglucomannan on the growth curve of Aspergillus fumigatus in a Sabourauddextrose broth supplemented with chloramphenicol and gentimycin. Thex-axis represents hours of culture. The y-axis represents density of theculture measured at an absorbance of 530 nm.

FIG. 3. shows the effects of adding a high concentration (500 μg/10 mlculture) of a mixture of 95.6% mineral clay and 4.4% β-1,3(4)glucan andglucomannan on the growth curve of Aspergillus fumigatus in a Sabourauddextrose broth supplemented with chloramphenicol and gentimycin. Thex-axis represents hours of culture. The y-axis represents density of theculture measured at an absorbance of 530 nm.

FIG. 4. shows effects of adding diatomaceous earth (50 μg/10 ml ofculture) on the growth curve of Aspergillus fumigatus in a Sabourauddextrose broth supplemented with chloramphenicol and gentimycin. Thex-axis represents hours of culture. The y-axis represents density of theculture measured at an absorbance of 530 nm.

FIG. 5. shows the effects of adding a three-way combination of 95.6%mineral clay plus 4.4% β-1,3(4)glucan and glucomannan (500 μg/10 mlculture) and diatomaceous earth (50 μg/10 ml culture) on A. fumigatusgrowth as described for FIGS. 2-4. The x-axis represents hours ofculture. The y-axis represents density of the culture measured at anabsorbance of 530 nm.

FIG. 6. shows the effects of adding β-1,3(4)-endoglucanohydrolase alone(100 μg/ml of culture medium) and a combination ofβ-1,3(4)-endoglucanohydrolase (100 μg/ml culture medium) and 100 μg/mlof the mixture used in Experiment 6 (FIG. 5: Combined ingredient) on A.fumigatus growth as described for FIG. 5. The y-axis represents thedensity of the culture measured at an absorbance of 530 nm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the novel discovery that a combinationof β-1,3(4)-endoglucanohydrolase, β-1,3(4)glucan, diatomaceous earth,mineral clay, and glucomannan effectively inhibit the growth ofpathogenic fungal species and thereby reduce or eliminate the direct orindirect negative consequences which accrue to the host mammalian oravian organism.

The β-1,3(4)-endoglucanohydrolase is from a commercial source and isproduced from submerged fermentation of a strain of Trichodermalongibrachiatum.

The diatomaceous earth is prepared by methods commonly known in the art.It is available as a commercially-available acid-washed, product with95% silica (SiO₂) and with its remaining components not assayed butconsisting primarily of ash (minerals) as defined by the Association ofAnalytical Chemists (AOAC, 2002).

The yeast cell wall extract is prepared by a method commonly known inthe art. It is a commercial source of β-1,3(4)glucan and glucomannanderived from primary inactivated yeast (Saccharomyces cerevisiae) withthe following chemical composition: Moisture 2-3% Dry matter 97-98%Proteins 14-17% Fats 20-22% Phosphorous 1-2% Mannans 22-24% β-1,3(4)glucan 24-26% Ash 3-5%

The mineral clays (aluminosilicates) used in this invention may befulfilled by any of a variety of commercially-available clays including,but not limited to, montmorillonite clay, bentonite and zeolite.

In a preferred embodiment of the invention,β-1,3(4)-endoglucanohydrolase, diatomaceous earth, yeast cell wallextract and mineral clay are combined at 0.05-3%, 1-40%, 1-20% and40-92%, respectively. In a preferred composition,β-1,3(4)-endoglucanohydrolase, diatomaceous earth, yeast cell wallextract and mineral clay are combined at 0.1-3%, 5-40%, 2-10% and40-80%, respectively. In an especially preferred embodiment of theinvention, β-1,3(4)-endoglucanohydrolase, diatomaceous earth, yeast cellwall extract and mineral clay are combined at 0.2-3%, 30-40%, 4-6% and50-65%, respectively. The preferred physical form of the invention is adry, free-flowing powder which is suitable for direct inclusion into afeed, food product or as a supplement to a total mixed ration or diet.

The compositions provided by the present invention may be incorporateddirectly into commercially-available feeds or food products or fed assupplements to commercially-available feeds or food products. Thecomposition contained in the present invention may be fed to anymammalian or avian species. The methods of the invention comprisereducing the growth and associated mycosis caused by enteric infectionsof pathogenic fungal organisms in the gut of mammalian and avianspecies. When incorporated directly into feeds, the present inventionmay be added to feeds in amounts ranging from 0.1 to 5 kg per ton offeed. In an especially preferred composition, the invention may be addedto feeds in amounts ranging from 1-2 kg per ton of feed.

The composition contained in the present invention may be added toanimal feedstuffs or to foods in amounts ranging from 0.0125% to 2% byweight of feed. In a preferred embodiment, the composition is added toanimal feedstuffs or to food in amounts from 0.0625% to 1% by weight offeed. In an especially preferred embodiment, the invention is added inamounts from 0.125% to 0.5% by weight of feed.

Alternatively, the composition contained in the present invention may befed directly to mammalian or avian species as a supplement in amounts0.016 grams/kg to 0.37 grams/kg of live body weight per day. In anespecially preferred embodiment, the invention may be provided tomammalian and avian species in amounts of 0.10 grams/kg to 0.20 grams/kgof body weight per day. One of skill and art can appreciate that theamount of the invention fed can vary depending upon the animal species,size of the animal and type of the feedstuff to which the invention isadded.

The novel methods of this invention comprise the ability of acombination of β-1,3 (4)-endoglucanohydrolase, diatomaceous earth, yeastcell wall extract and clay to inhibit the enteric growth and mycosiscaused by various pathogenic fungal genera which include, but are notlimited to, Aspergillus, Aureobasidium, Candida, Eurolium, Fusarium,Mucor, Penicillium and Rachiborskiomyces sp. The benefits resulting fromthe application of the invention to mammalian species include, but arenot limited to, reduced death losses, reduced incidence of mycoticabortion, reduced incidence of jejunal hemorrhage syndrome (dead gutsyndrome), reduced incidence of scouring (diarrhea), improved growthrate, improved efficiency of growth, improved milk production, improvedefficiency of milk production and reduced somatic cell counts in milkproducts (dairy animals). The benefits from the application of theinvention to avian species include, but are not limited to, reduceddeath losses, improved growth and egg production, improved fertility,and reduced incidence of enteric diseases.

EXAMPLES

The following are intended to be illustrative of the invention, and arenot to be considered restrictive of the scope of the invention asotherwise described herein.

Example 1

The following novel experiment documents the presence of mold spores orconidia in the feed, jejunal contents and jejunal wall of a Holsteindairy cow which died in 2002 from jejunal hemorrhage syndrome (dead gutsyndrome).

Samples of feed, jejunal contents and jejunal tissue were homogenized ina Polytron and serial dilutions (1 ml) of these samples were applied toa Petrifilm® mold count plate. The feed sample was centrifuged followinghomogenization to generate a particulate fraction and a solublefraction. The density of mold counts in each of these samples is shownin Table 1. TABLE 1 Density of colony forming units (cfu) in feed andtissue fractions of a cow which died from jejunal hemorrhage syndrome.Sample Colony forming units (cfu) feed (supernatant fraction)  4000colony forming units (cfu)/g feed feed (particulate fraction) 20000cfu/g feed jejunal contents of dead cow  100 cfu/ml gut contentsaffected jejunal tissue 11000 cfu/g tissue (wet weight)

The data indicate the presence of mold in feed and the gut. Of interest,the mold sample preferentially localized into the jejunal wall, acharacteristic of Aspergillus fumigatus. These data indicated potentialfor the fungal infection to underlie the etiology which led to death ofthe animal.

Example 2

The following experiment documents a novel discovery in which wedetermined that fungi can colonize the gut, invade the blood and producea mycotic condition which can result in jejunal hemorrhage, mycoticabortion and death of dairy animals. To complete this study, severalnovel steps were undertaken:

The sequence of Aspergillus fumigatus 18S small ribosomal subunit genewas determined from existing literature (Jaeger et al., 2000) and usedto design DNA primers for polymerase chain reaction (PCR) analysis ofthe presence of Aspergillus DNA in the gut, tissues and blood of cowsexhibiting mycotic abortion or which had died from jejunal hemorrhage.Two sets of primers were prepared: a primary pan-fungal set whichamplified all fungal DNA and a “nested set” which specifically-amplifiedand detected Aspergillus genera (Jaeger et al., 2000).

Additional primers (from the 18S gene) were designed for Sybr-Greenanalysis (real-time quantitative PCR) to allow for the determination ofthe mold DNA concentration (mold “burden”) in blood of cows which hadmycotic abortions or which had died from jejunal hemorrhage syndrome.

Using the DNA primers designed in Step 1 (above) we determined that cowsafflicted with jejunal hemorrhage syndrome or which displayed mycoticabortions exhibited high levels of Aspergillus mold counts in jejunalwall and blood. Using our novel real-time Sybr-Green quantificationprotocol (Step 2, above) we determined that the mold burden in cowswhich had either died from jejunal hemorrhage syndrome or which haddisplayed incidence of mycotic abortion were extremely high. Control(asymptomatic) cows did not harbor fungal DNA. Instead, via sequencing,we have detected other non-pathogenic fungal species (e.g.,Cladosporium) at low concentrations. This has led us to conclude thatlower levels of Aspergillus infection (mycosis) result in abortion(known as “mycotic abortion”) whereas exceedingly high levels result indeath of the infected animal. Whether or not death results from a directeffect of fungal infection or, instead, from secondary (indirect)bacterial infections (e.g., Clostridium sp.) has not been determined.

Example 3

The following novel experiment illustrates the ability of a mixture ofclay and β-1,3(4)glucan/glucomannan (95.6% and 4.4%, respectively) toinhibit the growth of Aspergillus fumigatus in culture. A. fumigatusculture was derived from a local corn grain sample and applied as astreak to a culture plate containing Sabouraud dextrose agar mediumsupplemented with chloramphenicol and gentimycin (to inhibit bacterialgrowth). Drops (50 μl) of sodium-aluminum silicate clay combined withβ-1,3(4)glucan and glucomannan (40 mg/ml: 95.6% clay, 4.4%β-1,3(4)glucan and glucomannan) were applied to the A. fumigatus streaksand the growth of the mold culture was evaluated following 42 hours ofculture at 27° C. The mold culture at 42 hours is shown as a zigzagpattern of white mold with spreading mycelia (see Appended FIG. 1).Drops of the clay/β-1,3(4)glucan and glucomannan product can be seenvisually as brown-colored areas on the culture dish. One such spot inFIG. 1 is indicated at the tip of a piece of white paper marked “I”.Application of the clay/β-1,3(4)glucan and glucomannan product to theculture clearly and effectively diminished growth of A. fumigatus.

Example 4

This novel experiment shows that additions of the mineral clay,β-1,3(4)glucan and glucomannan mixture (95.6% clay, 4.4% β-1,3(4)glucanand glucomannan) effectively inhibit the growth of Aspergillusfumigatus. The inhibition of fungal growth with these combinedingredients, represents a portion of the mechanism of action which wesubmit as a mechanism of action for products in the treatment andprevention of mycotic diseases in mammalian and avian species.

Aspergillus was inoculated into 10 ml of Sabouraud dextrose brothsupplemented with chloramphenicol and gentimycin (to inhibit bacterialgrowth). In addition, various amounts of a combination of mineralclay:β-1,3(4)glucan and glucomannan were added directly to cultures toestablish the effects of these compounds on the growth of theAspergillus culture. The density of cells was utilized as an index of A.fumigalus cell number and density was monitored using aspectrophotometer (wavelength was 530 nm).

In control cultures (i.e., A. fumigatus with no additions of the threecomponents of the invention), we typically observed a long lag phase(see appended FIG. 2) where little fungal growth occurred. This wasfollowed by a rapid, “log-phase” growth curve with maximum fungal celldensity being reached after several hours. When a combination of mineralclay:β-1,3(4)glucan and glucomannan product was added to the culture,the growth of the yeast culture was delayed (see appended FIGS. 2 and3). Specifically, addition of mineral clay:β-1,3(4)glucan andglucomannan mixture in combination, delayed entry of the A. fumigatusculture into the rapid log-phase growth. However, once A. fumigatusgrowth began, this product did not limit the total growth of theculture. The lowest effective dose of the clay: β-1,3(4)glucan andglucomannan combination was 100 μg/10 ml culture where a delay of 1-2hours in growth was observed (see appended FIG. 2). Higher levels of themineral clay: β-1,3(4)glucan and glucomannan mixture (e.g., 500 μg/10ml) delayed entry of the A. fumigatus into log-phase growth (seeappended FIG. 3).

Of interest, the transit time of digesta in an adult bovine animal is48-72 hours. The poorer growth conditions which A. fumigatus would findin the bovine digestive tract (i.e., due to competition with othermicrobial species, less growth substrate and less oxygen) would mostlikely alter its growth in such a manner that a delay in log-phasegrowth could result in loss of the infectious organism in the fecesbefore it has opportunity to rapidly proliferate. Hence, we propose thatthe delay in the log-phase fungal growth caused by the presence of a95.6% mineral clay with 4.4% β-1,3(4)glucan and glucomannan mixtureeffectively reduces the degree of colonization of the gut which may becaused by Aspergillus and other fungal genera and thereby reduces theharmful direct, and possibly indirect, effects of an Aspergillusinfection or infection by other pathogenic fungal species.

Example 5

This novel experiment documents the ability of diatomaceous earth toinhibit the growth of A. fumigatus in culture.

Similar to Example 4, diatomaceous earth was added to cultures of A.fumigatus which had been supplemented with chloramphenicol andgentimycin (to inhibit bacterial growth). A control sample was preparedto study fungal growth in the absence of diatomaceous earth. Inaddition, various levels of diatomaceous earth (5, 50, 250, 500, 1000and 5000 μg/10 ml culture) were added to A. fumigatus cultures todetermine its effects on fungal growth. Culture conditions wereidentical to those outlined in Example 4.

FIG. 4 (appended) documents the novel and surprising ability ofdiatomaceous earth to markedly reduce growth of a fungal culture. Thelowest effective dose at which diatomaceous earth inhibited fungalgrowth was 50 μg/10 ml of culture medium (FIG. 4, appended). Efficacywas also detected up to concentrations of 1000 μg/10 ml of culturemedium (data not included).

Example 6

This novel experiment documents the additive ability of a 3-waycombination of diatomaceous earth, mineral clay and β-1,3(4)glucan andglucomannan mixture to effectively inhibit fungal growth.

In this experiment, Aspergillus fumigatus was cultured as described inprevious examples. The effects of adding a mixture of all threeingredients on growth of A. fumigatus were studied. The mineral clay:β-1,3(4)glucan and glucomannan mixture delayed entry into log phasegrowth (as described in Examples 2-5). Diatomaceous clay (50 μg/10 ml ofculture) in combination with mineral clay: β-1,3(4)glucan andglucomannan mixture (500 μg/10 ml culture) inhibited growth of theAspergillus culture (i.e., a longer delay in entry into log-phasegrowth; see appended FIG. 5). Effects were greater than when productswere added alone.

Example 7

This novel experiment documents the ability ofβ-1,3(4)-endoglucanohydrolase, alone and in combination with the othercomponents of the invention, to markedly inhibit growth of A. fumigatusin liquid culture. In this experiment, the abilities ofβ-1,3(4)-endoglucanohydrolase alone (100 tμ/ml) and a combination ofβ-1,3(4)-endoglucanohydrolase (100 μg/ml) and a mixture of diatomaceousearth, mineral clay and β-1,3(4)glucan/glucomannan (100 μg/ml) werecombined and their effects on fungal growth (as described above) wereassessed. The β-1,3(4)-endoglucanohydrolase alone was unable to reducefungal growth (FIG. 6). Surprisingly, however, addition of this enzymeto the combination of diatomaceous earth, mineral clay andβ-1,3(4)glucan/glucomannan caused a marked reduction in fungal growth.Specifically, initiation of fungal growth was delayed from 4 hours toover 7 hours (FIG. 6).

Summary of Examples

These results show that the composition of the invention (i.e., mineralclay, yeast cell wall extract, diatomaceous earth andβ-1,3(4)-endoglucanohydrolase) is capable of a previously-undescribedeffect of inhibiting growth of pathogenic fungal species; species whichhave documented adverse effects on morbidities and mortalities ofmammalian and avian species. The combination of products reduces growthof pathogenic fungi in the gut of mammalian and domestic species andthereby prevents the invasion and colonization of the blood compartment(mycosis) and represents a mixture which is flowable in easilyincorporated into feed products and food products. The inventionspecifically prevents fungal-based septicemia and the deleterious directand indirect effects resulting thereof The present invention waseffective in achieving its inhibitory effects under growth conditionswhich might be found in mammalian and avian digestive systems wherenutrients, moisture, oxygen and elevated temperatures are provided bythe host.

The foregoing description of the preferred embodiment of the inventionhas been presented for purposes of illustration and description. It isnot intended to be exhaustive or to limit the invention to the preciseform disclosed. Obvious modifications or variations are possible inlight of the above illustrations. The embodiment was chosen anddescribed to provide the best illustration of the principles of theinvention and its practical application to thereby enable one ofordinary skill in the art to utilize the invention in variousembodiments and with modifications as are suited to the particular usecontemplated. All such modifications and variations are within the scopeof the invention as determined by the appended claims when interpretedin accordance with the breadth to which they are fairly, legally andequitably entitled.

1. A composition comprising a combination of β-glucans,β-1,3(4)-endoglucanohydrolase, calcined diatomaceous earth, a mineralclay, and glucomannan is admixed in foods and animal feeds therebyinhibiting fungal growth in the food, feedstuff or digesta of mammalianand avian species, thereby reducing susceptibility to a mycoticcolonization of the digestive tract and invasive mycoses.
 2. Thecomposition of claim 1, wherein the infecting fungal species includesone of the following fungal genera: Aspergillus, Aureobasidium, Candida,Eupenicillium, Eurotium, Fusarium, Mucor, Penicillium, Rachiborskiomyce.3. The composition of claim 1, wherein the infectious organism is amember of the Aspergillus genera.
 4. The composition of claim 1, whereinthe infectious organism is Aspergillus fumigatus.
 5. The composition ofclaim 1, wherein the mineral clay product is montmorillonite, bentonite,aluminosilicate, or zeolite clays, or mixtures thereof.
 6. Thecomposition of claim 1, wherein the β-1,3(4)-endoglucanohydrolase isproduced from submerged fermentation of Trichoderma longibrachialum. 7.The composition of claim 1, wherein the β-glucans and glucomannan arederived from boiling and enzyme autolysis of gram positive yeast cellwalls from the genera of Saccharomyces.
 8. The composition of claim 7,wherein the β-glucans and glucomannan are derived from boiling andenzyme autolysis of gram positive yeast cell walls from Saccharomycescerevisiae.
 9. The composition of claim 1, wherein the diatomaceousearth is calcined at a minimum temperature of 900° C.
 10. Thecomposition of claim 1, wherein the composition comprises between 15%and 40% diatomaceous earth, between 50% and 81% mineral clay, between1.0% and 5.0% β-glucans, between 0.05% and 3.0%β-1,3(4)-endoglucanohydrolase and between 1% and 8.0% glucomannan. 11.The composition of claim 1, wherein the composition comprises between20% and 30% diatomaceous earth, between 60% and 75% mineral clay,between 1.0% and 3.5% β-glucans, between 0.1% and 3.0%β-1,3(4)-endoglucanohydrolase and between 1.0% and 6.0% glucomannan. 12.The composition of claim 1, wherein the combination of diatomaceousearth, a mineral clay, β-1,3(4)-endoglucanohydrolase, β-glucan andglucomannan is admixed into foods or animal feedstuffs in aconcentration of between 0.0125% and 5% by weight for the purpose ofinhibiting fungal growth in feed, food or digesta.
 13. The compositionof claim 1, wherein said composition is admixed into a food or feedstuffand is subsequently fed to domestic livestock.
 14. The composition ofclaim 1, wherein said composition is admixed into a food or feedstuffand is subsequently feed to ruminant livestock.
 15. The composition ofclaim 1, whereas said composition reduces mycotic colonization of feeds,foods and the gastrointestinal tract.